This project has been concerned with two aspects of DNA replication, the biosynthesis of deoxyribose and the use of isolated nuclei as a means of identifying factors and mechanisms associated with replication control. As a model system, the allosteric properties of the ribonucleotide reductase of L. leichmannii are being investigated. This enzyme is a single polypeptide chain whose specificity for reduction of particular NTP's to the corresponding dNTP's is determined by a variety of nucleotide effectors whose binding constants for the single effector site have been determined. We will investigate the action of individual effectors by measuring effect of ligand saturation on rates of enzyme reaction with specific alkylating agents and on fluorescent and other reporter groups attached to the enzyme. To magnify effects, these studies will be carried out in the presence of limited concentrations of denaturants to partially destabilize enzyme conformation. Attempts will be made to mreasure substrate binding to the substrate site or sites by using effector and coenzyme analogs to obtain a complete system capable of substrate binding but not of reduction. This preparation will be used to determine existence of multiple substrate sites and site-site interaction if it exists. Isolated nuclei: From both HeLa cells and diploid human fibroblasts, isolated nuclei, and if possible, fragmented nuclei, will be used to detect and assay factors required for normal replication. The basic approach will invove supplementation of protein deficient preparations (from cells pretreated with cycloheximide) with fractions from actively replicating preparations. Activating factors that can be distinguished from extra DNA or from generalized nuclease action will be purified. The rifamycin derivative, AF/013 will be used to investigate the maturation of nascent DNA chains, particularly the intermediate sized fragments whose synthesis is stimulated by the drug.